MYCOBIOLOGY 2019, VOL. 47, NO. 4, 391–400 https://doi.org/10.1080/12298093.2019.1682449 RESEARCH ARTICLE A Survey of Termitomyces (Lyophyllaceae, Agaricales), Including a New Species, from a Subtropical Forest in Xishuangbanna, China Lei Yea,b,c , Samantha C. Karunarathnaa,b, Huli Lia,b,c, Jianchu Xua,b, Kevin D. Hydea,b,c,d and Peter E. Mortimera aKey Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China; bWorld Agroforestry Centre, East and Central Asia Office, Kunming, China; cCenter of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand; dMushroom Research Foundation, Chiang Mai, Thailand ABSTRACT ARTICLE HISTORY A survey of mushrooms was conducted in Xishuangbanna, Yunnan Province, China, in the Received 10 June 2019 rainy season (May to October) of 2012, 2013, and 2014, during which 16 specimens of Revised 10 September 2019 Termitomyces were collected. Preliminary macro- and micro-characteristics, together with ITS Accepted 10 September 2019 sequence data, showed that four of the specimens belonged to a new species (Termitomyces KEYWORDS fragilis), while the other 12 belonged to T. aurantiacus, T. eurrhizus, T. globules, T. microcarpus, Termitomyces; symbiosis; and T. bulborhizus. In this paper, T. fragilis is introduced as a species new to science based termites; wild on morphological characterization and phylogenetic analyses. Macro- and micro- morpho- edible mushrooms logical descriptions, color photographs and line drawings of the new species, and a phylo- genetic tree to show the placement of the new species are provided. T. fragilis is then compared with other closely related taxa in the genus Termitomyces. 1. Introduction tropical vegetation resources providing suitable habitats for a wide range of insects, and more spe- In 1942, R. Heim described specimens of a mush- cifically, termites [30]. Xishuangbanna is also home room genus that exhibited a close symbiotic relation- to a high diversity of Termitomyces species, which ship with termites (Isoptera) [1]. In recognition of are collected for consumption and for sale in mar- this termite-fungal mutualistic association, the genus kets, providing additional income to rural house- was named as Termitomyces [2–6]. Termitomyces are holds [31–33]. highly sought-after mushrooms, due to their high As a result of previous studies, more than 400 culinary value, providing many rural households with samples of Termitomyces have been deposited in dif- – additional income as well as nutrition [7 15]. ferent fungal herbaria in China [13], but sequence Various species of Termitomyces are widely dis- data, especially nuclear ribosomal internal tran- – tributed in Africa and Southeast Asia [16 19], and scribed spacer (ITS) gene, is largely unavailable in several taxa have also been recorded in Central GenBank, and there are few publications relating to America [20]. Molecular phylogenetic analysis this genus. Many of the past research studies inves- reveals that Termitomyces forms a monophyletic tigating Termitomyces have aimed to study their – clade in Agaricales [21 23], although genetically relationship with termites, ignoring the taxonomy they exhibit a degree of differentiation between Asia and phylogeny of this genus [22,34–39]. Recent and Africa [6]. The southern part of the Yangzi DNA-based studies of Termitomyces are limited; for River basin in China is rich in wild fungal resources example, in Asia, Sawhasan et al. published DNA such as Termitomyces [13,24–26]. In the southwest- data for 13 species of Termitomyces from Thailand, ern province of Yunnan, Termitomyces mushrooms and Adhikari and Durrieu published DNA data for are locally famed for their taste and association with five species of Termitomyces from Nepal, and termites. Locally they are known as “Jizong”, mean- Mossebo et al. published new taxa result inferred ing chicken-mushroom (due to the texture of the from combined nuclear ribosomal large subunit mushroom flesh), or “Yizong”, meaning ant-planted (nLSU) and mitochondrial small subunit ribosomal mushroom [25,27–29]. DNA (mtSSU-rDNA) sequences of Termitomyces Xishuangbanna is a tropical county located in [40–42]. There are, however, no further DNA-based south Yunnan Province, China, with abundant publications of Termitomyces in Asia. CONTACT Peter E. Mortimer [email protected] ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 392 L. YE ET AL. Based on the need for further research on the of collections; Lm ¼ mean basidiospore length over Termitomyces genus, we set out to study the com- a population of basidiospore; Wm ¼ mean basidio- position of Termitomyces species in a subtropical spore width over a population of basidiospore; rainforest in Xishuangbanna, Yunnan Province, Q¼“Length/width ratio” (l/W) of a basidiospore in China. In this study, we provide molecular based side view; Qm ¼ average Q of all basidio- descriptions of the new species; morphological spores measured. descriptions of all the collected specimens; and a record of the related habitats. 2.3. DNA extraction, PCR, and sequencing Genomic DNA was extracted from three dried 2. Materials and methods specimens of the new species using a Biospin 2.1. Specimen collection and macro Fungus Genomic DNA Extraction Kit (Bioer morphological descriptions Technology Co., Ltd., Hangzhou, P. R. China). The All 16 specimens were collected during the rainy ITS gene region of the new species was amplified. m season (May to October), from 2012 to 2014, in Amplification was performed in 25 l volumes con- m m Mengsong, Xishuangbanna, Yunnan Province, taining 1.0 l template DNA, 9.5 l double distilled m China. The vegetation cover at the collection sites water, 1.0 l of each primer (ITS1/ITS4) [49] and m  was dominated by Camellia sinensis var. assamica, 12.5 lof2 power Taq PCR Master Mix (A pre- Castanopsis mekongensis, Schima wallichii, and mix and ready to use solution, including 0.1 Units/ m m Quercus acutissima. Specimens were photographed l Taq DNA Polymerase, 500 M dNTP Mixture – in situ, then gathered and wrapped in aluminum each (dATP, dCTP, dGTP, dTTP), 20 mM Tris HCl foil, or kept separately in a collecting box in order (pH8.3), 100 mM KCl, 3 mM MgCl2, stabilizer and to avoid mixing or crushing of specimens, and enhancer. The reaction was carried out with 35 finally returned to the laboratory for further analysis cycles under the following conditions: denaturation (95 C, 30 s), annealing (52 C, 30 s), extension and characterization. Odor and color changes upon bruising were recorded at the time of collection. (72 C, 1 min), and final extension (72 C, 10 min). Description of macro-characteristics, chemical test- The primers used for sequencing the whole ITS ing, and further photographing of fresh samples region were the same as those used in White et al. were carried out soon after returning to the field [49]. Amplified products were confirmed with 1% station each day. This was done in accordance with agarose gel electrophoresis stained with ethidium the methodology described by Largent [43,44]. The bromide. The amplified PCR fragments were sent to Kornerup and Wanscher method was used to a commercial sequencing provider (Beijing Bai Mai describe color terms, where a specific code is Hui Kang Biological Engineering Technology Co., assigned to an observed color (chromotaxy) [45]. Beijing, P.R. China). The newly generated sequence Specimens were dried below 40 C in a food drier, data of the new species were deposited in GenBank. sealed in plastic bags, and deposited in the Kunming Institute of Botany herbarium (HKAS). 2.4. Sequence alignment and Facesoffungi numbers and Index Fungorum num- phylogenetic analyses bers were obtained as detailed in Jayasiri et al. and Index Fungorum [46,47]. The taxon information and GenBank accession num- bers used in the molecular analyses are listed in Table 1. All the available reliable nrITS sequences 2.2. Micro morphological description were retrieved from the GenBank. Sequences for each Micro-morphological features were documented by species were aligned using Clustal X [50]. Alignments examining dried specimens following the methods were manually adjusted to allow for maximum of Largent [48]. For micro-morphological examin- sequence similarity. Gaps were treated as missing ation, sections were cut with a razor blade from data. Unweighted maximum parsimony (MP) ana- dried specimens and mounted on slides in 5﹪ lysis was performed using PAUP 4.0b10 [51]. Trees KOH and Congo red, and then observed, measured were inferred using the heuristic search option with and illustrated using a compound microscope (Zeiss TBR branch swapping and 1000 random sequence Axioskop 40). In the description of the basidio- additions. Max trees were unlimited, branches of zero spores, “n” indicates the number of basidiospores length were collapsed, and all multiple parsimonious (20 basidiospores per collection) which were meas- trees were saved. Clade stability of the trees resulting ured; [a/b/c] in the taxonomic description refer to from the parsimony analyses were assessed by boot- the following: a ¼ number of basidiospores meas- strap analysis with 1000 replicates, each with 10 repli- ured, b ¼ number of fruiting bodies and c ¼ number cates of random stepwise addition of taxa [52]. MYCOBIOLOGY 393 Bayesian phylogenetic analyses were performed with proposed to be a new species to science; of these, all MrBayes 3.2.2 [53–55]. Four Markov chains were run four specimens were shown to be the same species, for 1,000,000 generations and sampled every 100th as described below. generation, resulting in 1000 trees. Those trees Termitomyces fragilis L. Ye, Karun, J.C.